Use of active substances in lycium ruthenicum murr

Abstract

The application discloses application of an active substance in Lycium ruthenicum Murr, relates to the technical field of natural product separation, and application of the active substance in Lycium ruthenicum Murr in sugar metabolism and lipid metabolism, including application in preparation of products for treating and / or preventing diseases related to sugar or lipid metabolism disorder and application in preparation of at least one of PPAR gamma antagonists, C / EBP alpha antagonists, FAS inhibitors and ACC inhibitors. It is found that the active substance norpetanin separated from Lycium ruthenicum Murr has a function of reducing sugar and reducing lipid.

Description

Technical Field

[0001] This invention relates to the field of natural product separation technology, and in particular to the application of an active substance in black goji berries. Background Technology

[0002] Black goji berries are a medicinal and edible plant that has been widely cultivated in recent years in areas such as the Qaidam Basin in Qinghai Province. They have high nutritional and edible value, containing various nutrients such as protein, vitamins, and minerals. The fruit is also rich in anthocyanins, which have antioxidant and anti-allergic properties, enhance immunity, and improve sleep.

[0003] Studies have shown that black goji berries contain a variety of bioactive components. Their ripe berries are rich in anthocyanins and other polyphenols, which have antioxidant properties and potential preventative and therapeutic effects on cardiovascular diseases. Among these, polyphenols, including anthocyanins and flavonoids, exhibit various biological activities such as anti-inflammation, cardiovascular protection, and anti-tumor effects through their strong antioxidant free radical scavenging properties.

[0004] Current research on the active substances in black goji berries is relatively weak, and further research is needed to discover, isolate, and extract its active ingredients. If more in-depth and detailed research can be conducted on the new chemical components and their pharmacological effects, as well as to explore their active mechanisms, it is hoped that more safe and effective natural plant-derived drugs that can treat diseases can be developed. Summary of the Invention

[0005] The purpose of this invention is to apply an active substance in black goji berries. For the first time, an active substance, norpetanin, in black goji berries has been discovered, which has the effects of lowering blood sugar and lowering blood lipids.

[0006] The technical solution adopted in this invention is:

[0007] An application of an active substance in black goji berries, the application including the use of the active substance and its pharmaceutically acceptable salts, hydrates or solvates in improving glucose metabolism and / or lipid metabolism, wherein the active substance is a compound of general formula I or its various optical isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates:

[0008]

[0009] Furthermore, the application in glucose metabolism is its use in the preparation of products for the treatment and / or prevention of diseases related to glucose metabolism disorders.

[0010] Furthermore, the product is a product for improving insulin resistance.

[0011] Furthermore, the application in lipid metabolism is its use in the preparation of products for the treatment and / or prevention of diseases related to lipid metabolism disorders.

[0012] Furthermore, the product is one that has lipid-lowering effects or inhibits adipocyte differentiation.

[0013] Furthermore, the product is a product that inhibits the accumulation of intracellular lipid droplets and / or the production of intracellular lipids.

[0014] Furthermore, the product is at least one of the following: products for treating and / or preventing obesity, hypertension, hyperlipidemia, cardiovascular disease, and metabolic syndrome-related diseases.

[0015] Furthermore, the application also includes its use in the preparation of at least one of PPARγ antagonists, C / EBPα antagonists, FAS inhibitors, and ACC inhibitors.

[0016] Furthermore, the active substance and its pharmaceutically acceptable salts, hydrates, or solvates comprise compounds as shown in Formula II or Formula III:

[0017]

[0018] Furthermore, the ratio of the compounds represented by Formula II and Formula III in the active substance is 3:1.

[0019] The active substance or its solvate, hydrate, pharmaceutically acceptable salt or eutectic pharmaceutical composition of the present invention may contain pharmaceutically acceptable excipients.

[0020] In this invention, "pharmaceutically acceptable" means any substance that does not interfere with the bioactivity of the active ingredient and is non-toxic to the host to which it is given.

[0021] The pharmaceutically acceptable excipients described in this invention refer to all additional materials in a drug other than the active pharmaceutical ingredient (API). Excipients should possess the following properties: (1) non-toxic to the human body and with virtually no side effects; (2) chemically stable and not easily affected by temperature, pH, storage time, etc.; (3) without incompatibility with the API and without affecting the efficacy and quality control of the API; and (4) not interacting with packaging materials. In this invention, excipients include, but are not limited to, fillers (diluents), lubricants (flow aids or anti-adhesion agents), dispersants, wetting agents, adhesives, regulators, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrups, gum arabic, gelatin, sorbitol, astragalus gum, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, or hydroxypropyl methyl cellulose), gelatin paste, syrup, starch paste, or polyvinylpyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, dicalcium phosphate, precipitated calcium carbonate, etc.), sorbitol, or glycine, etc.; lubricants include micronized silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, etc.). The ingredients include: pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.; polyvinylpyrrolidone or microcrystalline cellulose, etc.; humectants include sodium dodecyl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutylbenzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% chlorobutanol, etc.; regulators include hydrochloric acid, citric acid, potassium hydroxide (sodium), sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate-80, sorbitan, prorhonic acid F-68, lecithin, soybean lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc. The term "pharmaceuticalally acceptable salt" refers to a salt formed by the compounds of this invention with an acid or base that is suitable for use as a medicine. The acids and bases mentioned above are Lewis acids and bases in a broad sense. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and benzenesulfonic acid; as well as acidic amino acids such as aspartic acid and glutamic acid.

[0022] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0023] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in the dosage forms of capsules, tablets, and pills.

[0024] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0025] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0026] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0027] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0028] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0029] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0030] The compounds of this invention can also be used in injectable formulations. The injectable formulation is selected from liquid injections (water injections), sterile powders for injection (powder injections), or tablets for injection (referring to molded or machine-compressed tablets made by aseptic methods, dissolved in water for injection before use, for subcutaneous or intramuscular injection).

[0031] In addition to the aforementioned compounds, the injectable powder also contains at least an excipient. The excipients described in this invention are components intentionally added to the drug. They should not possess pharmacological properties in the amount used; however, excipients may contribute to drug processing, dissolution or release, drug delivery via targeted administration routes, or contribute to stability.

[0032] The beneficial effects of this invention are:

[0033] This invention is the first to obtain an active substance, norpetanin, from black goji berries, and discovers that the active substance norpetanin has the effect of lowering blood sugar and lipids.

[0034] The numbers marked on the structural formulas of the compounds in this invention are only for the purpose of explaining the structure of the compounds. Attached Figure Description

[0035] Figure 1 High-resolution mass spectrum of norpetanin;

[0036] Figure 2 norpetanin 1 H NMR spectrum;

[0037] Figure 3 norpetanin 13 C NMR spectrum;

[0038] Figure 4 HMBC diagram of norpetanin;

[0039] Figure 5 HSQC diagram of norpetanin;

[0040] Figure 6 COSY plot of norpetanin;

[0041] Figure 7 DEPT plot of norpetanin;

[0042] Figure 8 UV absorption spectrum of norpetanin;

[0043] Figure 9 Figure showing the effect of norpetanin treatment on 2-NBDG uptake in 3T3-L1 adipocytes as detected by FCM method;

[0044] Figure 10 The effect of norpetanin on the expression of p-PI3K and p-AKt in 3T3-L1 adipocytes;

[0045] Figure 11 Effect of norpetanin on lipid droplet accumulation in 3T3-L1 cells (100×);

[0046] Figure 12 The effect of norpetanin on TG content in 3T3-L1 cells;

[0047] Figure 13 Figure showing the effect of norpetanin on the expression of adipogenic transcription factors in 3T3-L1 cells;

[0048] Figure 14 Figure showing the effect of norpetanin on the expression of lipid metabolism-related proteins in 3T3-L1 cells. Detailed Implementation

[0049] The technical solution of the present invention is described clearly and completely below. Obviously, the embodiments described herein are only a part of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0050] Example 1

[0051] The active substance is a compound with the structure shown in general formulas II and III, or its optical isomers, crystal forms, pharmaceutically acceptable salts, hydrates, or solvates:

[0052]

[0053] The nuclear magnetic resonance data of active substances in black goji berries are shown in Table 1.

[0054] Table 1. Nuclear magnetic resonance data of active substances

[0055]

[0056] Active substance: Yellow powder, ESI-MS m / z: 809 [MH] - The main component has the molecular formula C 36 H 42 O 21 The m / z value was determined to be 809.2140 [MH] by HR-ESI-MS. - The calculated value is 809.2141, with an unsaturation degree of 19. Its NMR spectrum shows paired signals of varying intensities, indicating the presence of tautomerism. For ease of structural analysis, the NMR signals of the predominantly trans-tautomers are preferentially used for structural elucidation. As shown in Table 1, 1 H NMR(DMSO-d6 / CF3COOD(9:1),600MHZ):δ H The signals at 7.53 (2H, d, J = 8.5 Hz, H-2”” / H-6””), 6.79 (2H, d, J = 8.5 Hz, H-3”” / H-5””), 7.53 (1H, d, J = 15.9 Hz, H-7””), and 6.35 (1H, d, J = 15.9 Hz, H-8””) show a characteristic set of trans-para acyl groups, and the signal at δH 7.53 (1H, s, H-4) shows an aromatic or alkene proton singlet state. H The signals at 6.54 (1H, d, J = 1.7 Hz, H-6) and 6.36 (1H, d, J = 1.7 Hz, H-8) indicate a set of 1,3,4,5-tetrasubstituted benzene rings, δ H Signals at 5.09 (1H(d), J = 7.3 Hz, H⁻¹') and 4.82 (1H”) indicate two β-glucanose anomeric protons, δ H The signal at 4.61 (1H, br s, H-1”) indicates an α-rhamnosyl anisoproton, δ H The signal at 4.82 (1H, t-like, J = 9.6 Hz, H-4”) shows an acylated methine triplet state, and the upper field region shows a rhamnose methyl doublet state. 13C10 NMR (DMSO-d6 / CF3COOD(9:1), 600MHZ): shows a total of 36 carbon resonances, including a set of trans-p-acyl carbons, and 18 carbon signals attributable to the three sugar moieties, while the remaining 9 unattributed resonance carbons originate from the glycoside ligand nucleus. The above NMR characteristics are generally similar to those of petanin, the main difference being the absence of the ring B signal in the anthocyanin nucleus in this compound. This allows us to infer that the ring B moiety must have been oxidized and degraded to yield the specific 3,5,7-trioxy-substituted coumarin nucleus. From H-4 to C-2 [δ] C 157.6(s)], C-5[δC 154.5(s))] and C-9[δ C 152.1(s)], from H-6 and H-8 to C-10[δ C 103.1(s)] and from H-1' to C-3[δ C The HMBC correlation of 138.0(s)], such as Figure 4 As shown, this inference is confirmed. A careful analysis of HSQC, HMBC, and... 1 H, 1 The H-COSY correlation further validated the obtained structure, such as Figure 2 , 4 As shown in Figure 6, the new compound is isolated as a combination of inseparable tautomers (trans:cis ≈ 3:1). Therefore, the structure of the active substance is shown in Formulas II and III, and it is named norpetanin. To our knowledge, the active substance norpetanin represents a rare class of cyclic β-anthocyanins.

[0057] Its DEPT plot, UV absorption spectrum, and high-resolution mass spectrum are as follows: Figure 7 , 8 As shown in Figure 1.

[0058] Example 2

[0059] Effects of norpetanin on glucose metabolism:

[0060] 1. Experimental Materials and Instruments

[0061] 1.1 Materials and Reagents

[0062] Test substance: Norpetanin, prepared in the laboratory.

[0063] 3T3-L1 mouse embryonic fibroblasts were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The materials and reagents used in the experiment are shown in Table 2.

[0064] Table 2 Materials and Reagents

[0065]

[0066]

[0067] Main experimental reagents

[0068] (1) Preparation of 2-NBDG

[0069] 2-NBDG powder was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted with sugar-free DMEM to a final concentration of 100 μM before use.

[0070] (2) The Western blotting working solution is prepared as follows:

[0071] a. 10% SDS

[0072] Weigh 10g of SDS, add 100mL of ultrapure water, dissolve in a 50℃ water bath, and store at room temperature. If precipitation occurs, it can still be used after dissolving in a water bath.

[0073] b. SDS-PAGE electrophoresis buffer (5×)

[0074] Weigh 15.1g Tris, 94.0g Glycine, and 5.0g SDS, dissolve in ultrapure water, and bring the volume to 100mL. Store at room temperature. Before use, dilute the 5× electrophoresis buffer to 1×. It can be reused 2-3 times. Store at room temperature.

[0075] c. Transfer buffer (10×)

[0076] Weigh 15.15g Tris and 72.0g Glycine, and dilute to 1× transfer buffer before use. Method: Measure 50mL of 10× transfer buffer, add 100mL of methanol, and make up to 500mL with ultrapure water, i.e., 10× transfer buffer: methanol: water = 1:2:7. It can be reused 3-5 times and stored in a refrigerator at 4℃.

[0077] d.TBS(10×)

[0078] Weigh 12.12g Tris and 40.03g NaCl, dissolve in ultrapure water, adjust pH to 7.6 with HCl (add approximately 3.5mL HCl), bring volume to 500mL, and store at 4℃.

[0079] 1.2 Instruments and Equipment

[0080] The main instruments and equipment used in this experiment are shown in Table 3.

[0081] Table 3 Instruments and Equipment

[0082]

[0083]

[0084] 2 Experimental Methods

[0085] 2.1 Establishment of the 3T3-L1 adipocyte model

[0086] (1) In vitro culture and passage of 3T3-L1 cells

[0087] 3T3-L1 cells were cultured in DMEM high-glucose medium containing a mixture of 10% FBS and 1% penicillin-streptomycin. The cell culture incubator was maintained at 37°C, 95% humidity, and 5% CO2. Under normal culture conditions, 3T3-L1 cells needed to be passaged every 3 days. When the cell density reached 85%–90% or higher, the original culture medium was first aspirated using a pipette pump, and the cells were washed 1–2 times with warmed PBS. Immediately afterwards, 850 μL of trypsin digestion solution was added, and the culture dish was gently agitated to ensure the digestion solution evenly covered the entire bottom. Under a microscope, when the cells became rounded and detached from the bottom of the dish, fresh culture medium was added, and the cells were repeatedly pipetted to completely detach from the bottom of the dish and disperse into single cells. The cell suspension was passaged into new culture dishes at a 1:5 ratio, and appropriate amounts of DMEM and FBS were added before the cells were placed in the cell culture incubator for further culture.

[0088] (2) Cryopreservation and thawing of 3T3-L1 cells

[0089] Cell cryopreservation: Select cells in the logarithmic growth phase, rapidly digest the cells, and centrifuge the digested cell suspension at 1000g for 5 minutes at room temperature to obtain cell clusters. Add 1.2 mL of pre-prepared cell cryopreservation solution (DMEM:FBS:DMSO = 5:4:1) to each cryopreservation tube, and label the tube wall with (cell type, cell passage number, number of cells to be cryopreserved, and cryopreservation time) for easy reference. Finally, incubate at 4℃ for 10 minutes, at -20℃ for 1 hour, and at -80℃ overnight. The next day, store in liquid nitrogen. Cell thawing: Quickly remove the cryopreserved cell lines from the liquid nitrogen tank and rapidly thaw them in a 37℃ water bath. After the cells are completely thawed, quickly transfer them to culture medium for culture. After the cells adhere to the culture medium (generally 6-8 hours), replace with fresh culture medium.

[0090] (3) Counting of 3T3-L1 cells

[0091] The commonly used tool for cell counting is a hemocytometer. Each hemocytometer consists of two H-shaped grooves forming a counting cell 0.10 mm high. The counting chamber contains nine large squares, with the four corner squares used for cell counting. After determining the cell count, the cell density is adjusted according to experimental needs. The general principle for counting is to record the top squares but not the bottom, and the left squares but not the right. Adjust the cell counting density according to experimental requirements, record the total number of cells in the four corner squares, and finally calculate the cell count using the formula:

[0092] Number of cells = Total number of cells in 4 cells / 4 × 10 4 ×Dilution factor

[0093] (4) Establishment of an insulin resistance model

[0094] The "cocktail" method was used to induce differentiation of 3T3-L1 cells. The induction of differentiation was carried out as follows: 3T3-L1 cells in good condition were seeded on culture plates at a density of 5 × 10⁶ cells / cm². 4 Cells were cultured at a density of approximately 85%–90% in high-glucose DMEM medium containing 10% FBS. The medium was then changed, and after two days of contact inhibition, the complete culture medium was discarded. A culture medium containing 10 μg / mL Insulin, 0.5 mM IBMX, and 1 μM Dex was added, and the cells were cultured for 2 days (recorded as day 0, Induction I). The medium was then changed to contain 10 μg / mL Insulin (Induction II) and cultured for another 2 days. Finally, the medium was changed to normal medium and cultured for another 4 days, with the medium changed every other day. On day 8 after induced differentiation and maturation, 1 μM Dex was added to the cell slurry to establish an IR model.

[0095] 2.2 Effect of norpetanin on 2-NBDG uptake in 3T3-L1 cells

[0096] 3T3-L1 preadipocytes were divided into 5×10 4 Cells were seeded at a density of [number] cells / mL in 12-well plates. Once the cell density reached over 80%, differentiation was induced. On day 8 after induced differentiation and maturation, 1 μM Dex was added to the cell slurry to establish an IR model. Three groups were set up: a normal group, a model group, and a drug-treated group. Except for the normal group, which was cultured in complete medium, the other groups were cultured in 1 μM Dex. In the drug-treated groups, 10 μM of the test substance was added and the cells were cultured for 48 h. The culture medium was then aspirated, the cells were washed once with DPBS, and 500 μL of trypsin was added for digestion at 37°C for 1 min. 2 mL of DPBS was added, and the cells were mixed thoroughly and centrifuged at 1000g for 6 min. The supernatant was discarded, and 1 mL of sugar-free medium containing 10 μM 2-NBDG was added to each well. The cells were incubated at 37°C for 30 min, and the fluorescence intensity was detected by flow cytometry at wavelengths of 488 nm and 515-545 nm.

[0097] 2.3 Effect of norpetanin on AKt phosphorylation in 3T3-L1 cells

[0098] 3T3-L1 preadipocytes were divided into 5×10 4 Cells were seeded at a density of 10 cells / mL into 6-well plates. When the cell density reached more than 80%, differentiation was induced. On day 8 after induced differentiation and maturation, 1 μM DEx was added to the cell culture medium to establish an IR model. Normal, model, and drug-treated groups were set up. Except for the normal group, which was cultured in complete medium, the other groups were cultured in 1 μM DEx. The drug-treated groups were each cultured with 10 μM norpetanin for 48 h. After culturing, the culture medium was aspirated, cells were collected, and the expression levels of PI3K and AKt proteins were detected by Western blotting.

[0099] 2.4 Western blot analysis

[0100] Protein extraction was performed on the cell samples obtained after processing. The specific operation method is as follows: (1) Mix RIPA lysis buffer and PMSF at a ratio of 100:1 (if analyzing phosphorylated proteins, phosphatase inhibitors need to be added) to prepare cell lysis buffer. Wash the cells in the 6-well plate three times with cold PBS, add 100 μL of cell lysis buffer to each well, place on ice for 5 min for lysis, then scrape the cells with a cell scraper and collect them into a 1.5 mL centrifuge tube, and place it in a 4℃ freezer for 30 min for complete lysis. Finally, centrifuge the liver tissue homogenate and cell lysis buffer at 12000g, 4℃, for 15 min, and collect the supernatant protein into a new EP tube.

[0101] (2) BCA method for protein content determination: Take an appropriate amount of protein sample. According to the instructions of the BCA protein quantification kit, dilute the protein standard with PBS buffer to 0.5 mg / mL. Mix reagent A and reagent B in a 50:1 ratio to prepare the working solution. Add the standard and PBS to a 96-well plate as shown in Table 4, 20 μL per well. Generally, three replicates are set for each concentration to plot the standard curve. Add 1 μL of protein sample to the 96-well plate and add PBS to make up to 20 μL. Add 200 μL of BCA working solution to each well and mix thoroughly. Incubate the above samples in a microplate at 37°C for 30 min with a shaker. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample according to the standard curve. Heat the prepared protein sample at 100°C for 10 min to completely denature the protein. Before loading the protein, centrifuge and mix the sample thoroughly.

[0102] Table 4. Protein Quantification by BCA Method

[0103] 1 0 20 200 0 2 1 19 200 0.5 3 2 18 200 1 4 4 16 200 2 5 8 12 200 4 6 12 8 200 6 7 16 4 200 8 8 20 0 200 10

[0104] (3) SDS-PAGE electrophoresis: Select an appropriate separating gel concentration based on the molecular weight of the target protein (as shown in Table 5), prepare a suitable solution system (lower gel), and allow the lower gel to solidify completely at room temperature for at least 40 minutes. Then prepare a 5% stacking gel (upper gel) and insert a comb of appropriate size. After the stacking gel solidifies, place the gel plate in the electrophoresis tank, fill the space between the two plates with electrophoresis buffer, remove the comb, load the protein sample and marker, and run the gel. Electrophoresis conditions: S1 stage, 80V, 30min; S2 stage, 120V, approximately 60–90min, until the bromophenol blue in the buffer reaches the bottom of the separating gel, then stop the electrophoresis.

[0105] (4) Transfer: Immediately after protein electrophoresis, transfer the membrane. Cut the target protein according to its molecular weight based on the marker bands. Cut the PVDF membrane to the same size as the gel. Activate the cut membrane in methanol solution for 1 minute, then place it in the transfer buffer. Clamp the electrophoresis clamps in the following order: transfer clamp blackboard - filter paper - PVDF gel - filter paper - transfer clamp whiteboard, with the blackboard facing the black side of the tank. Place the electrophoresis tank in the tank and fill it completely with transfer buffer. After powering on, place the transfer apparatus in an ice-water mixture. The transfer conditions are a constant current of 250 mA and a time of 90 minutes.

[0106] Table 5. Optimal separation range of SDS-PAGE separating gels at different concentrations.

[0107] 6 50-150 8 30-90 10 20-80 12 12-60 15 10-40

[0108] (5) Blocking: After the transfer is completed, the PVDF membrane is quickly and gently removed from the transfer plate with tweezers and rinsed in TBST buffer for 5 min. Then, it is immediately blocked in TBST containing 5% skim milk powder on a shaker at room temperature for 1 h.

[0109] (6) Primary antibody incubation: After the blocking is completed, the PVDF membrane is placed in the diluted primary antibody solution and incubated overnight on a horizontal shaker at 4°C.

[0110] (7) Secondary antibody incubation: After the primary antibody incubation, wash the membrane three times with TBST, each time for 8 min. Then place the PVDF membrane in a solution containing HRP-labeled mouse or rabbit secondary antibody and incubate slowly at room temperature for 1 h.

[0111] (8) Development: After the secondary antibody incubation, the membrane was washed three times with TBST, each time for 8 minutes. After washing, the mixed ECL developing solution was dropped onto the PVDF membrane and photographed using a gel imaging system.

[0112] (9) Results analysis: The gray values ​​of the target protein and the internal reference protein were quantitatively analyzed using ImageJ software. The relative expression level of the protein was the ratio of the target protein to the internal reference protein (the ratio of phosphorylated to non-phosphorylated protein).

[0113] 2.5 Statistical Analysis

[0114] GraphPad Prism7 software was used for plotting and statistical analysis. Independent samples t-tests were used to compare the means between two groups, and one-way ANOVA was used to compare the means among multiple groups. Statistical data are expressed as mean ± standard deviation (x ± s). * or # indicates P < 0.05, indicating statistical significance, and ** or ## indicates P < 0.01, indicating highly statistically significant difference.

[0115] 3 Experimental Results

[0116] 3.1 Effect of norpetanin on 2-NBDG uptake in 3T3-L1 cells

[0117] like Figure 9 As shown, the uptake of glucose by 3T3-L1 adipocytes was detected using the fluorescently labeled glucose analog 2-NBDG. The fluorescent probe 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-ylamino]-2-deoxyglucose) is a fluorescent analog of 2-deoxyglucose that specifically binds to intracellular glucose, exhibiting high sensitivity. Normally, the fluorescence intensity of 2-NBDG in insulin-resistant cells or organisms is extremely weak, at background levels, indicating a weak glucose uptake capacity. Experimental results showed that the uptake capacity of 2-NBDG in the normal group was higher than that in the insulin-resistant model group. The glucose uptake capacity of cells in the model group was weaker, showing a significant difference compared to the glucose uptake capacity of cells in the normal group after insulin stimulation. Intervention with the active substance norpetanin promoted the uptake of 2-NBDG by adipocytes under insulin stimulation, suggesting that norpetanin has the potential to improve insulin resistance.

[0118] Note: Compared with the normal group # P<0.05, ## P < 0.01; compared with the model group, *P < 0.05, **P < 0.01

[0119] 3.2 Effects of norpetanin on PI3K and AKt phosphorylation in 3T3-L1 cells

[0120] AKt, also known as protein kinase B (PKB), is a serine / threonine protein kinase that plays a crucial role in various cellular processes, including glucose metabolism, apoptosis, cell proliferation, and cell transport. During glucose metabolism, activated AKt activates various downstream factors in the insulin signaling pathway, such as enzymes, kinases, and transcription factors, through phosphorylation, thereby regulating cellular function and insulin signal transduction. Typically, activated AKt activates its downstream phosphatidylinositol kinase 3 (PI3K), promoting the translocation of glucose transporters from the cytoplasm to the cell membrane, accelerating glucose uptake and utilization, and thus playing a role in insulin signal transduction. This experiment investigated the effects of norpetanin, an active substance from black goji berries, on the expression of p-AKt and p-PI3K in cells to examine whether it promotes glucose uptake and utilization, enhances insulin signal transduction, and improves insulin resistance. The experimental results are as follows: Figure 10 As shown, compared with the normal group, the expression levels of p-AKt and p-PI3K proteins were decreased in the model group. Treatment with the active substance norpetanin can increase the expression levels of p-AKt and p-PI3K in 3T3-L1 adipocytes, thereby promoting glucose uptake and enhancing insulin sensitivity in 3T3-L1 adipocytes.

[0121] 3.3 Summary

[0122] An insulin resistance model was established using 3T3-L1 adipocytes to investigate the hypoglycemic effect of norpetanin, an active substance from black goji berries. FCM was used to detect the uptake of glucose analogs by the compound in adipocytes under insulin resistance. Western blotting was used to detect the phosphorylation levels of PI3K and AKt in 3T3-L1 adipocytes. The results showed that intervention with norpetanin promoted the uptake of 2-NBDG by adipocytes under insulin stimulation, demonstrating its potential to improve insulin resistance. Western blotting results also showed that norpetanin increased the expression levels of p-PI3K and p-AKt in 3T3-L1 adipocytes.

[0123] In this study, the active substance norpetanin from black goji berries increased the expression levels of p-AKt and p-PI3K in 3T3-L1 adipocytes, suggesting that norpetanin can promote glucose uptake by insulin target tissues, improve insulin sensitivity, and has the potential to improve insulin resistance. This provides more scientific evidence for the development and utilization of black goji berries to treat type II diabetes.

[0124] Example 3

[0125] Effects of norpetanin on lipid metabolism:

[0126] 1. Experimental Materials and Instruments

[0127] 1.1 Materials and Reagents

[0128] Test substance: Norpetanin, which was isolated and prepared in the laboratory.

[0129] 3T3-L1 mouse embryonic fibroblasts were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The materials and reagents used in the experiment are shown in Table 6.

[0130] Table 6 Materials and Reagents

[0131]

[0132]

[0133] 1.2 Instruments and Equipment

[0134] The main instruments and equipment used in this experiment are shown in Table 7.

[0135] Table 7 Instruments and Equipment

[0136] Haier HYCD-205 Refrigerator / Freezer Qingdao Haier Co., Ltd. Micro 21R high-speed refrigerated centrifuge Thermo Fisher Scientific Inc. LS-750 liquid nitrogen tank Chengdu Jinfeng Liquid Nitrogen Container Co., Ltd., Sichuan LX71 Inverted Microscope OLYMPUS BE-9008 Micro-plate Thermostatic Oscillator Shanghai Yanhe Instrument Equipment Co., Ltd. Tanon 520 Gel Imaging System Shanghai Tianneng Technology Co., Ltd. Multifunctional ELISA reader Molecular Devices Corporation ultrasonic cleaner Dingshanhu Instrument Factory, Jiangsu Province Electrophoresis apparatus Bio-Rad HH-2 Digital Display Constant Temperature Water Bath Jintan City Kexing Instrument Factory Pipettes of different sizes Eppendorf LDZM-80KCS Vertical Pressure Steam Sterilizer Shanghai Shenan Medical Equipment Factory SW-CJ-2FD Type Double-Person Single-Sided Cleanroom Workbench Suzhou Purification Equipment Co., Ltd. Constant temperature carbon dioxide incubator SANYO

[0137] 2 Experimental Methods

[0138] 2.1 Establishment of the 3T3-L1 adipocyte model

[0139] (1) In vitro culture and passage of 3T3-L1 cells

[0140] 3T3-L1 cells were cultured in DMEM high-glucose medium containing a mixture of 10% FBS and 1% penicillin-streptomycin. The cell culture incubator was maintained at 37°C, 95% humidity, and 5% CO2. Under normal culture conditions, 3T3-L1 cells needed to be passaged every 3 days. When the cell density reached 85%–90% or higher, the original culture medium was first aspirated using a pipette pump, and the cells were washed 1–2 times with warmed PBS. Immediately afterwards, 850 μL of trypsin digestion solution was added, and the culture dish was gently agitated to ensure the digestion solution evenly covered the entire bottom. Under a microscope, when the cells became rounded and detached from the bottom of the dish, fresh culture medium was added, and the cells were repeatedly pipetted to completely detach from the bottom of the dish and disperse into single cells. The cell suspension was passaged into new culture dishes at a 1:5 ratio, and appropriate amounts of DMEM and FBS were added before the cells were placed in the cell culture incubator for further culture.

[0141] (2) Cryopreservation and thawing of 3T3-L1 cells

[0142] Cell cryopreservation: Select cells in the logarithmic growth phase, rapidly digest the cells, and centrifuge the digested cell suspension at 1000g for 5 minutes at room temperature to obtain cell clusters. Add 1.2 mL of pre-prepared cell cryopreservation solution (DMEM:FBS:DMSO = 5:4:1) to each cryopreservation tube, and label the tube wall with (cell type, cell passage number, number of cells to be cryopreserved, and cryopreservation time) for easy reference. Finally, incubate at 4℃ for 10 minutes, at -20℃ for 1 hour, and at -80℃ overnight. The next day, store in liquid nitrogen. Cell thawing: Quickly remove the cryopreserved cell lines from the liquid nitrogen tank and rapidly thaw them in a 37℃ water bath. After the cells are completely thawed, quickly transfer them to culture medium for culture. After the cells adhere to the culture medium (generally 6-8 hours), replace with fresh culture medium.

[0143] (3) Counting of 3T3-L1 cells

[0144] The commonly used tool for cell counting is a hemocytometer. Each hemocytometer consists of two H-shaped grooves forming a counting cell 0.10 mm high. The counting chamber contains nine large squares, with the four corner squares used for cell counting. After determining the cell count, the cell density is adjusted according to experimental needs. The general principle for counting is to record the top squares but not the bottom, and the left squares but not the right. Adjust the cell counting density according to experimental requirements, record the total number of cells in the four corner squares, and finally calculate the cell count using the formula:

[0145] Number of cells = Total number of cells in 4 cells / 4 × 10 4 ×Dilution factor

[0146] (4) 3T3-L1 cell differentiation induction

[0147] The "cocktail" method was used to induce differentiation of 3T3-L1 cells. The induction of differentiation was carried out as follows: 3T3-L1 cells in good condition were seeded on culture plates at a density of 5 × 10⁶ cells / cm². 4 Cells were cultured in high-glucose DMEM medium containing 10% FBS until the cell density reached approximately 85%–90%. The medium was then changed, and after two days of contact inhibition, the complete culture medium was discarded. A culture medium containing 10 μg / mL Insulin, 0.5 mM IBMX, and 1 μM Dex was added, and the cells were cultured for 2 days (recorded as day 0, Induction I). The medium was then changed to contain 10 μg / mL Insulin (Induction II) and cultured for another 2 days. Finally, the medium was changed to normal culture medium and cultured for another 4 days, with the medium changed every other day. On day 8 of induction, more than 85% of the cells exhibited the morphology of mature adipocytes, with lipid droplets of varying sizes arranged in a "ring-like" pattern.

[0148] 2.2 Oil Red O staining

[0149] Healthy 3T3-L1 cells were seeded into 6-well plates at a density of 5 × 10⁶ cells / well. 4 Cells were cultured in high-glucose DMEM medium containing 10% FBS until the cell density reached approximately 85%–90%. The medium was then changed, and after two days of contact inhibition, the complete culture medium was discarded. A culture medium containing 10 μg / mL Insulin, 0.5 mM IBMX, and 1 μM MDex was added, and the cells were cultured for 2 days (recorded as day 0, Induction I). The medium was then changed to contain 10 μg / mL Insulin (Induction II) and cultured for another 2 days. Afterward, the medium was changed to normal medium with 10 μM norpetanin for co-incubation, with the medium changed every other day. After induction, cells were fixed with 4% neutral formaldehyde for 30 min. After fixation, Oil Red O working solution was added to stain the cell surface, and the cells were incubated in the dark for 60 min. After staining, the cells were washed with 70% ethanol to remove excess dye, and then washed 3–4 times with ultrapure water. Finally, the cells were observed and photographed under a microscope.

[0150] 2.3 Determination of TG content

[0151] 3T3-L1 cells were used at a rate of 5 × 10⁻⁶ 4 Cells were seeded at a density of 100 cells / mL in 6-well plates. Differentiation was induced when the cell density reached approximately 85%–90%. TG content was measured on day 8 of induction, using the following specific method:

[0152] (1) Cell pretreatment: On day 8 of induction, the cell culture medium was aspirated, the cells were washed twice with cold PBS, and then trypsin digestion solution was added to digest the cells.

[0153] (2) Cell collection: After cell digestion, the cells were resuspended in PBS and centrifuged at 1000g for 5 minutes to collect the cell pellet.

[0154] (3) Ultrasonic disruption: Add an appropriate amount of PBS to the collected precipitate and then ultrasonically disrupt it (3 min).

[0155] (4) Measurement: Add 2 μL of cell lysate suspension to each well of a 96-well plate, 2 μL of distilled water to the blank wells, and 2 μL of standard to the standard wells. Then add 200 μL of the assay solution to each well, mix well, incubate at 37°C for 10 min, and read the absorbance at 510 nm. Determine the protein concentration in the sample using the BCA method, perform calibration, and finally calculate the TG content according to the formula.

[0156] TG content = (OD sample - OD blank) / (OD calibration sample - OD blank) × calibrator concentration (mM) / protein concentration of the sample to be tested (gprot / L)

[0157] 2.4 Western blot analysis

[0158] The obtained cell samples were then subjected to protein extraction, and the specific procedure was as follows:

[0159] (1) Mix RIPA lysis buffer and PMSF at a ratio of 100:1 (if analyzing phosphorylated proteins, a phosphatase inhibitor needs to be added) to prepare cell lysis buffer. Wash the cells in the 6-well plate three times with cold PBS, add 100 μL of cell lysis buffer to each well, and lyse on ice for 5 min. Then scrape the cells with a cell scraper and collect them into 1.5 mL centrifuge tubes, and place them in a 4°C freezer for 30 min to fully lyse. Finally, centrifuge the liver tissue homogenate and cell lysis buffer at 12000g, 4°C, for 15 min, and collect the supernatant protein into a new EP tube.

[0160] (2) BCA method for protein content determination: Take an appropriate amount of protein sample. According to the instructions of the BCA protein quantification kit, dilute the protein standard with PBS buffer to 0.5 mg / mL. Mix reagent A and reagent B in a 50:1 ratio to prepare the working solution. Add the standard and PBS to a 96-well plate as shown in Table 8, 20 μL per well. Generally, three replicates are set for each concentration to plot the standard curve. Add 1 μL of protein sample to the 96-well plate and add PBS to make up to 20 μL. Add 200 μL of BCA working solution to each well and mix thoroughly. Incubate the above samples in a microplate at 37°C for 30 min with a shaker. Measure the absorbance at 562 nm using a microplate reader and calculate the protein concentration of the sample according to the standard curve. Heat the prepared protein sample at 100°C for 10 min to completely denature the protein. Before loading the protein, centrifuge and mix the samples thoroughly.

[0161] Table 8. Protein Quantification Table Using BCA Method

[0162] 1 0 20 200 0 2 1 19 200 0.5 3 2 18 200 1 4 4 16 200 2 5 8 12 200 4 6 12 8 200 6 7 16 4 200 8 8 20 0 200 10

[0163] (3) SDS-PAGE electrophoresis: Select an appropriate separating gel concentration based on the molecular weight of the target protein (as shown in Table 9), prepare a suitable solution system (lower gel), and allow the lower gel to solidify completely at room temperature for at least 40 minutes. Then prepare a 5% stacking gel (upper gel) and insert a comb of appropriate size. After the stacking gel solidifies, place the gel plate in the electrophoresis tank, fill the space between the two plates with electrophoresis buffer, remove the comb, load the protein sample and marker, and run the gel. Electrophoresis conditions: S1 stage, 80V, 30min; S2 stage, 120V, approximately 60–90min, until the bromophenol blue in the buffer reaches the bottom of the separating gel, then stop the electrophoresis.

[0164] (4) Transfer: Immediately after protein electrophoresis, transfer the membrane. Cut the target protein according to its molecular weight based on the marker bands. Cut the PVDF membrane to the same size as the gel. Activate the cut membrane in methanol solution for 1 minute, then place it in the transfer buffer. Clamp the electrophoresis clamps in the following order: transfer clamp blackboard - filter paper - PVDF gel - filter paper - transfer clamp whiteboard, with the blackboard facing the black side of the tank. Place the electrophoresis tank in the tank and fill it completely with transfer buffer. After powering on, place the transfer apparatus in an ice-water mixture. The transfer conditions are a constant current of 250 mA and a time of 90 minutes.

[0165] Table 9. Optimal separation range of SDS-PAGE separating gels at different concentrations.

[0166] 6 50-150 8 30-90 10 20-80 12 12-60 15 10-40

[0167] (5) Blocking: After the transfer is completed, the PVDF membrane is quickly and gently removed from the transfer plate with tweezers and rinsed in TBST buffer for 5 min. Then, it is immediately blocked in TBST containing 5% skim milk powder on a shaker at room temperature for 1 h.

[0168] (6) Primary antibody incubation: After the blocking is completed, the PVDF membrane is placed in the diluted primary antibody solution and incubated overnight on a horizontal shaker at 4°C.

[0169] (7) Secondary antibody incubation: After the primary antibody incubation, wash the membrane three times with TBST, each time for 8 min. Then place the PVDF membrane in a solution containing HRP-labeled mouse or rabbit secondary antibody and incubate slowly at room temperature for 1 h.

[0170] (8) Development: After the secondary antibody incubation, the membrane was washed three times with TBST, each time for 8 minutes. After washing, the mixed ECL developing solution was dropped onto the PVDF membrane and photographed using a gel imaging system.

[0171] (9) Results analysis: The gray values ​​of the target protein and the internal reference protein were quantitatively analyzed using ImageJ software. The relative expression level of the protein was the ratio of the target protein to the internal reference protein (the ratio of phosphorylated to non-phosphorylated protein).

[0172] 2.5 Statistical Analysis

[0173] GraphPad Prism7 software was used for plotting and statistical analysis. Independent samples t-tests were used to compare the means between two groups, and one-way ANOVA was used to compare the means among multiple groups. Statistical data are expressed as mean ± standard deviation (x ± s). * or # indicates P < 0.05, indicating statistical significance, and ** or ## indicates P < 0.01, indicating highly statistically significant difference.

[0174] 3 Experimental Results

[0175] 3.1 Effects of norpetanin on lipid droplet accumulation in 3T3-L1 cells:

[0176] The effect of norpetanin on lipid droplet accumulation in 3T3-L1 cells was observed by Oil Red O staining, and the results are as follows: Figure 11 As shown, undifferentiated cells show no accumulation of lipid droplets, while differentiated cells contain a large number of lipid droplets. After treatment with norpetanin, the number of intracellular lipid droplets was significantly reduced.

[0177] 3.2 Effect of norpetanin on lipid content in 3T3-L1 cells:

[0178] The effect of norpetanin on TG content is as follows: Figure 12 As shown, compared with undifferentiated cells, differentiated cells had significantly increased intracellular TG content (P<0.01). Compared with differentiated cells, norpetanin treatment resulted in decreased TG content, and the differences were highly significant (P<0.01).

[0179] 3.3 Effects of norpetanin on the expression of lipid metabolism proteins in 3T3-L1 cells:

[0180] (1) Effect of norpetanin on the expression of adipogenic transcription factors in 3T3-L1 cells

[0181] The effect of norpetanin on the expression of adipogenic transcription factors in 3T3-L1 cells was analyzed by Western blot, and the results are as follows: Figure 13 As shown, the expression levels of PPARγ and C / EBPα proteins were low in undifferentiated 3T3-L1 cells, while the expression levels of PPARγ and C / EBPα proteins were high in differentiated cells. Compared with the differentiation group, norpetanin treatment could reduce the protein expression levels of PPARγ and C / EBPα transcription factors to some extent.

[0182] (2) Effects of norpetanin on the expression of lipidogenesis-related proteins in 3T3-L1 cells

[0183] The effect of norpetanin on the expression levels of lipid production-related proteins in 3T3-L1 cells was analyzed by Western blot, and the results are as follows: Figure 14As shown, the expression levels of FAS and ACC proteins were low in undifferentiated 3T3-L1 cells. However, the expression levels of FAS and ACC proteins were significantly increased in induced differentiated cells. Compared with the differentiated group, treatment with the compound significantly reduced the expression levels of FAS and ACC proteins. Based on these results, it was found that norpetanin reduces the accumulation of intracellular lipid droplets by inhibiting the expression of adipogenic transcription factors and lipidogenesis-related proteins.

[0184] 3.4 Summary

[0185] The effects of norpetanin on lipid production in 3T3-L1 cells were investigated at the cellular level. The differentiation of 3T3-L1 cells from preadipocytes into mature adipocytes is regulated by transcription factors and lipoproteins, and cell morphology also changes, eventually resulting in a "ring-like" appearance. Norpetanin can inhibit the accumulation of lipid droplets in 3T3-L1 cells and reduce intracellular triglyceride (TG) levels. Our results also indicate that norpetanin can inhibit the differentiation of 3T3-L1 adipocytes and reduce intracellular lipid droplet accumulation by suppressing the expression levels of transcription factors such as PPARγ and C / EBPα. Simultaneously, it improves cellular lipid metabolism by inhibiting the expression levels of FAS and ACC proteins, thereby suppressing lipid production.

Claims

1. An application of an active substance in black goji berries, characterized in that, The application is the use of the active substance or a pharmaceutically acceptable salt thereof in the preparation of products that improve glucose metabolism and / or lipid metabolism; the active substance has the structure of compounds as shown in Formulas II and III: ， The ratio of the compounds represented by Formula II and Formula III in the active substance is 3:

1.

2. The application according to claim 1, characterized in that, The product in question is for improving insulin resistance.

3. The application according to claim 1, characterized in that, The product in question has lipid-lowering effects or inhibits adipocyte differentiation.

4. The application according to claim 1, characterized in that, The product is designed to inhibit the accumulation of intracellular lipid droplets and / or the production of intracellular lipids.

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